Method for protection against genotoxic mutagenesis

ABSTRACT

A method and pharmaceutical for protecting against mutational damage in mammalian cells, irrespective of the nature of the mutagenic event or source of radiational or chemical insult or the like.

This is a divisional of application Ser. No. 08/121,946, filed Sep. 13,1993, now U.S. Pat. No. 5,567,686 which is a continuation-in-part ofapplication Ser. No. 07/851,210, filed Mar. 13, 1992, which issued asU.S. Pat. No. 5,488,042 on Jan. 30, 1996.

The present invention is generally directed to a method for protectingagainst genomic destabilization in mammalian cells from chemical orradiational mutagenic events and the like. More particularly, theinvention is concerned with mutation reduction through use ofS-ω-(ω-aminoalkylamino)alkyl dihydrogen phosphorothioates and theircorresponding metabolites.

Classic somatic mutation models of aging hold that the aging function isthe result of an accumulation, over time, of mutational events innuclear DNA; see, Kirkwood, Mutat. Res., Vol. 219, pp. 1-7 (1988),and/or mitochondrial DNA (mtDNA); see Linnane, et al, Mutat. Res. Vol.275, pp. 195-208 (1992). With respect to the contribution of mtDNAmutations to the phenotype of aging, the central premise is thataccumulation of random mutations in the cellular population is a majorcontributor to the gradual loss of cellular bioenergy capacity withintissues and organs, and that general senescence and diseases of agingare associated therewith.

Deletions of mtDNA were previously thought to occur only in individualswith neuromuscular disease. However, one particular deletion (mtDNA⁴⁹⁷⁷) accumulates with age primarily in non-dividing muscle and braincells. Consistent with the contribution of mtDNA to aging is that thegenome of this organelle appears especially sensitive to endogenous andenvironmental mutagens, given the lack of protective histones. It ispostulated that deleted mtDNA and DNA fragments may be further degradedor translocated from the mitochondria to the nucleus, a routesubstantiated by observations of inserted mtDNA sequences into nuclearDNA. Thus, it is speculated that fragments of migrating mtDNA may changethe information content and expression of certain nuclear genes. Suchgenomic destabilization may thereby promote aging and carcinogenicprocesses.

Age-dependent genomic alterations have also been observed in the nuclearDNA of dividing cells. Genomic destabilization is observed through theincidence of tumorigenic mutations that strike genes involved in thecontrol of cell proliferation, i.e., the protooncogenes and tumorsuppressor genes. See, Mutat. Res., Vol. 275, pp. 113-114 (1992). Theprinciple of chemoprevention is the reduction in incidence of mutagenicevents, thus preventing the onset of the carcinogenic process.

Mutagenesis, whether mitochondrial or nuclear in nature, is widelythought to be the result of the effect of reduced, reactive oxygenspecies and associated free radicals. Mitochondrial DNA is continuallyexposed to such oxy-radicals. The age-dependent decline in thecapability and capacity of mitochondria to dispose of these reactivespecies eventually render mtDNA more vulnerable to mutagenic eventsduring the aging process. Through a variety of proposed mechanisms, freeradicals, whether generated by radiation or during normal cellrespiration, have been shown in the prior art to induce a multitude ofdifferent DNA lesions in mammalian tissues, as well as in bacteria, andhave also been implicated in carcinogenic processes. See, Mutat. Res.,Vol. 269, pp. 193-200 (1992).

Reactive oxygen species and related free radicals may be generated withequal effect through a variety of exogenous (environmental) orendogenous agents, the result of chemical or radiational insult and thelike. Regardless of the origin or cellular mechanism, these mutagenicevents are expressed through genome destabilization and eventualmutagenesis. Ionizing radiation is often employed in laboratory studiesas a surrogate for other various environmental mutagenic agents. Thepropriety of such an assumption has been demonstrated in vitro usingstock cultures of selected hamster cell lines which exhibited identicalmutagenesis at the hypoxanthine-guanine phosphoribosyl transferase(HPRT) locus exposed to either ionizing radiation orcis-diaminedichloroplatinum (II). See, Grdina et al, Cancer Research,Vol. 46, pp. 1132-1135 (1986); and Grdina et al, Int. J. RadiationOncology Biol. Phys., Vol. 12, pp. 1475-1478 (1986).

The prior art is concerned with protecting against the genotoxic effectsof radiation by the S-ω-(ω-aminoalkylamino)alkyl dihydrogenphosphorothioates and has focused on the pre-irradiation effect ofdosages on amelioration of radiation's lethal effects with noappreciation for the anti-mutagenic, but only mutagenic effects. Inprior art uses, it was required to administer maximum tolerated levelsof the drugs prior to radiation exposure. Such requirements have limitedthe effectiveness of these agents because, when administered at therequired maximum tolerated dose, they are debilitating causing fever,chills, rash, hypotension, nausea and vomiting. It is conventionallyaccepted that the drugs must be administered prior to radiation exposurewhich heretofore has precluded their use for individuals accidentallyexposed to radiation.

Since 1949, the status of the prior art dictates that, in order for theradioprotective drug to be effective, it must be present beforeradiation exposure. The conventional understanding is also that thedisulfide form of radioprotectors is incapable of providing protection.In drugs such as WR-2721 the level of protection is proportional to theamount of the drug administered. The prior art also teaches there arepotential mutational properties of these agents which must be avoided.In particular, it has been suggested that one such agent in this classof phosphorothioates identified as S-2(3-aminopropylamino) ethylphosphorothioic acid (also known as "WR-2721"), by way of intracellularreactions, can lead to the conversion of cytosine moieties in DNA touracil. The result of use of WR-2721 can then be a mutagenic reaction innormal tissue.

These above enumerated concerns, along with conventional wisdom existingsince as long ago as 1949, have prevailed and have discouragedinvestigation into the potential of phosphorothioates and relatedaminothiol compounds as chemopreventative agents.

Radioprotection is distinguished from chemoprevention in that the formerrefers to protection against cell killing by irradiation and the latterrefers to protection against mutagenic and related carcinogenicprocesses. Phosphorothioates and related compounds, when employed asradioprotectors, are operationally defined as materials which canprotect against genotoxic damage induced by known mutagens andcarcinogens occurring as a result of ionizing radiation administeredafter ingestion of the chemical agent or drugs. The accepted protectivemechanisms of action of these drugs include: the scavenging of freeradicals produced as a result of the radiolysis of cellular water(presumably, free radical damage to DNA); the repair of chemical lesionsvia hydrogen atom donation; and the induction of cellular hypoxia. Thedeleterious effects of radiation occur via the deposition of energy inless than 10⁻¹² sec, while the relaxation of ionizations and excitationsoccur in less than 10⁻² sec. Damage to DNA, which leads to celllethality, is completed between 10⁻⁷ and 10⁻³ sec. These models areconsistent with the failure to demonstrate protection against celllethality by the phosphorothioates and related aminothiols when they areadministered immediately following radiation exposure.

In 1985 it was reported that a free thiol designated 2-(aminopropyl)amino! ethanethiol could protect against somatic mutationsat the hypoxanthine-guanine phosphoribosyl transferase locus in culturedrodent cells (designated V79), even if it were administered 3 hfollowing irradiation. These in vitro results relating to postirradiation exposure and protection by this agent against mutagenesiswere extended in 1989 to include protection against fission-spectrumneutrons. The extreme toxicity of this agent precluded its testing underin vivo conditions to ascertain the actual anti-mutagenic effect in amammal. In 1987 the drug cysteamine was tested as an antimutagen, but noprotective effects were observed unless it was present duringirradiation (administered prior to).

The problem of genome instability and subsequent mutagenesis isassociated both with endogenous and environmental mutagenic agents,including cosmic radiation, ultra violet light, radiation from nuclearreactors and war-released materials, and radiation from diagnostic andtherapeutic sources. The development of mutations and relatedcarcinogenic and aging processes arising from these and like radiationsources are well-documented and proven to be major health risks to thepopulation as a whole, as well as to high-risk groups employed in thenuclear power industry, military, and patients receiving diagnostic andtherapeutic radiation treatments. Likewise, mutagenic events originatefrom a variety of chemical and chemotherapeutic agents.

There exists a need for a method for protecting against mutationsirrespective of the source of mutagenic event or insult which will beamenable to pre- and/or post-radiation administration and which will beeffective at relatively low non-toxic concentrations so as to allow usein mammals and also allow for multiple, as well as single,administrations.

Accordingly, it is an object of the present invention to provide a novelmethod and substance for reducing mutations of mammal cells, includinghumans, exposed to radiation or chemical insult and like mutagenicevents.

It is another object of this invention to provide a method of andcompositions for protection against mutagenesis, irrespective of thesource of mutagenic event or insult, such that genome stabilization isprovided aid that aging and carcinogenic processes are inhibited.

It is another object of the invention to provide an improved method foruse of aminothiols and associated metabolites which diminish mutation ofboth cancerous and normal cells exposed to radiation or chemotherapy andthe like, whether administered before or after therapy.

It is an additional object of the invention to provide a method usingS-ω-(ω-aminoalkylamino)alkyl dihydrogen phosphorothioates to protectagainst initial mutagenic events irrespective of their source or natureand promote genome stabilization, such that subsequent mutagenesis andloss of genetic information is prevented.

It is still another object of the invention to provide a class ofaminothiol agents which metabolize in vivo to produce free sulfhydrylgroups and disulfides for protection against mutagenesis in mammaliancells.

It is a further object of the invention to provide a therapeutic routeby which an aminothiol and/or aminodisulfide metabolite of aphosphorothioate agent is utilized to provide protection againstmutagenic events and subsequent mutagenesis.

It is another object of this invention to provide a method for use of anantimutagenic agent to modulate cellular enzymatic processes, stabilizegenomic material, prevent loss of cell function and genetic information,and increase the efficiency of and time available for cell repairprocesses.

It is still another object of this invention to provide a method for usein vivo of an antimutagenic agent to enhance the fidelity of mutationalrepair through delay of cell cycle progression or related cellularmechanisms.

It is a further object of this invention to provide a method for in vivouse of compositions which are both reactive toward the deleteriousformation of free radical species by exogenous or endogenous sources andmitigate the mutational damage induced thereby, thus reducing theaccumulation of genetic mutations as manifested through aging andcarcinogenic processes.

These and other objects of the present invention will become apparentfrom consideration of the following description of preferredembodiments, examples, claims, and the drawings described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the performance of S-2-(3-aminopropylamino)ethylphosphorothioic acid (also identified as WR-2721) to protectagainst radiation-induced mutagenesis when administered to animalseither 30 min before, immediately after, or 3 h following irradiation.A. No treatment; B. WR2721, 400 mg/kg; C. WR2721, 400 mg/kg (3 times);D. Irradiation only; E. WR2721 before Irradiation; F. WR2721 afterIrradiation; G. WR2721 3 hr after Irradiation; and H. WR2721 3,24,48 hrafter Irradiation. Error bars represent one standard error of the mean;

FIG. 2 demonstrates the performance at low concentrations ofS-2-(3-aminopropylamino) ethylphosphorothioic acid (i.e., WR-2721) inthe range of from 400 mg/kg to 10 mg/kg. A. No treatment; B. WR2721, 400mg/kg; C. Irradiation only; D. WR2721, 40 mg/kg, before Irradiation; E.WR721, 200 mg/kg, before Irradiation; F. WR2721, 100 mg/kg, beforeIrradiation; G. WR721, 50 mg/kg, before Irradiation; and H. WR2721, 25mg/kg, before Irradiation; and I. WR2721, 10 mg/kg, before Irradiation.Error bars represent one standard error of the mean;

FIG. 3 demonstrates the relationship between the concentration of 2-(aminopropyl) amino! ethanethiol (i.e., WR-1065) and its protectiveability against radiation-induced (⁶⁰ Co Gamma-rays, 750 cGy)mutagenesis using CHO-AA8 cells (irradiated only (O); treated (▪). Eacherror bar is one standard error of the mean;

FIG. 4 demonstrates the effect of concentration of 2- (aminopropyl)amino! ethanethiol (i.e., WR-1065) on its protective ability againstradiation-induced (⁶⁰ Co Gamma-rays 750 cGy) lethality. Each error baris one standard error of the mean;

FIG. 5A, FIG. 5B and FIG. 5C. FIG. 5A demonstrates the effect ofcellular levels of 2- (aminopropyl) amino! ethanethiol (i.e., WR-1065)and its disulfide (i.e., WRSS) on the protection against cell killing inFIG. 5B and protection against mutagenesis in FIG. 5C followingirradiation with 150 cGy of fission-spectrum neutrons. Each error bar isone standard error of the mean;

FIG. 6A and FIG. 6B. FIG. 6A shows the structure of the disulfide form(designated WR-33278) of 2- (aminopropyl) amino! ethanethiol (designatedWR-1065) compared to the polyamine spermine (FIG. 6B);

FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E, FIG. 7F, FIG. 7G, FIG. 7H,FIG. 7I and FIG. 7J show the chemical structures of thephosphorothioates/aminothiols used;

FIG. 8 demonstrates the effectiveness under in vitro conditions of 3-(2-mercaptoethyl) amino! propionamide p-toluenesulfonate (designatedWR-2529); S-1-(aminoethyl) phosphorothioic acid (designated WR-638); S-2-(3-methylaminopropyl amino ethyl! phosphorothioate (designatedWR-3689), S-1-(2hydroxy-3-amino) propyl phosphorothioic acid (designated(WR-77913); and 2- 3-methylamino) propylamino! ethanethiol (designatedWR-255592) in protecting against radiation-induced mutagenesis A.Irradiation only; B. p529 Before and During; C. WR2529 After; D. WR638Before and During; E. WR638 After; F. WR3689 Before and During; G.WR3689 After; H. WR77913 Before and During; I. WR77913 after; J. WR55591Before and During; K. MEA Before and During; and L. MEA After. Theseresults are shown as a function of administration either 30 min beforeor immediately after irradiation with 150 cGy of fission-spectrumneutrons. Each error bar is one standard error of the mean; and

FIG. 9 demonstrates the effectiveness, under in vivo conditions, ofS-2-(3-aminopropylamino) ethyl phosphorothioic acid (WR-2721);S-1-(aminoethyl) phosphorothioic acid (WR-638); S- 2-(3-methylaminopropyl) aminoethyl! phosphorothioate acid (WR-3689);S-2-(4-aminobutylamino) ethylphosphorothioic acid (WR-2822);S-2-(5-aminopentylamino) ethyl phosphorothioic aid (WR-2823); 1-3-(3-aminopropyl) thiazolidin-2-Y1!-D-gluco-1,2,3,4,5-pentane-pentoldihydrochloride (WR-255709), in protecting against radiation-inducedmutagenesis as a function of administration either 30 min before orimmediately after irradiation of B6CF, mice with 150 cGy offission-spectrum neutrons A. 750 cGy, ⁶⁰ Co Gamma-rays; B. WR2721, 400mg/kg, before Irradiation (pooled); C. WR638 520 mg/kg before;Irradiation; D. WR3689 690 mg/kg before Irradiation; E. WR822 195 mg/kgbefore Irradiation; F. WR2823 200 mg/kg before Irradiation; G. WR255709300 mg/kg, before Irradiation; H. WR2721 400 mg/kg before Irradiation;I. WR721 400 mg/kg after Irradiation (pooled); J. WR638 520 mg/kg afterIrradiation; K. WR822 195 mg/kg after Irradiation; L. WR2823 200 mg/kgafter Irradiation; and M. WR2721 400 mg/kg after Irradiation.

FIG. 10A, FIG. 10B, FIG. 10C, FIG. 10D, FIG. 10E, FIG. 10F, FIG. 10G,FIG. 10H, FIG. 10I, FIG. 10J, FIG. 10K, FIG. 10L, FIG. 10M, FIG. 10N,FIG. 10O, FIG. 10P and FIG. 10Q demonstrate the inhibitition oftopoisomerase IIα activity in CHO K1 cells by the administration ofWR-1065, as either thiol- or disulfide-mediated.

FIG. 10A summarizes the effects of WR-1065 and radiation on theactivities of Topo I and IIα in K1 CHO cells, as determined by DNArelaxation and unknotting assays, respectively. Comparisons were made tothe corresponding untreated control groups using Student's two-tailed ttest. Comparisons not significant, p≧0.386, except as noted. Topo I andTopo IIα expressed in units/jig protein (mean ±S.D. of fourexperiments). Topo IIα in K1 cells +WR-1065, -γ-ray, significantdifference at p=0.019. Topo IIα in K1 cells+WR-1065, +γ-ray, suggestivedifference at p=0.061.

FIG. 10B summarizes the effects of WR-1065 and radiation on the proteinlevels of Topo IIα in K1 CHO cells, as determined by immunoblottingusing an anti-Topo II specific antibody. Comparisons were made to thecorresponding untreated control groups using Student's two-tailed ttest. All comparisons not significant, p>0.300. Results expressed asmean ±S.D of at least three experiments.

FIG. 10C shows survival curves for K1 CHO cells irradiated with 50-kVpx-rays. Cells were either treated with 4 mM WR-1065 (▪) or untreated(). Experimental points represent the mean of three experiments; errorbars represent the standard error of the mean. Survival curve parameterswere determined by using a computer-fitted least-squares regressionmodel.

FIG. 10D and FIG. 10E show, respectively, Topo Ilα and topo I activityin nuclear extracts from untreated and WR-1065-treated K1 cells. Nuclearextracts containing the following amounts of protein were assayed fortopo IIα-mediated unknotting and topo-I-mediated relaxing activities, asdescribed herein: FIG. 10D, lane 1, 80 ng; lane 2, 40 ng; lane 3, 20 ng;lane 4, 10 ng; lane 5, 5 ng; FIG. 10E, lane 1, 100 ng; lane 2, 30 ng;lane 3, 10 ng; lane 4, 3 ng; lane 5, 1 ng; (-), no nuclear extract. Thisis a representative experiment. Data from four such experiments wereused to determine the mean activities.

FIG. 10F results from immunoblot analysis of topo IIα levels in nuclearextracts from untreated and WR-1065-treated K1 cells. Logarithmicallygrowing cells were washed twice by centrifugation at 1000 x g for 5 minin PBS containing protease inhibitors and extracts. Nuclear proteinswere subjected to gel electrophoresis through an 8% SDS-polyacrylamidegel and transferred to nitrocellulose. Blots were incubated withanti-topo II antibody. The molecular weights shown on the right ordinateare those of topo IIα (MW 170,000) and its proteolytic products.Prestained standards with their molecular weights in thousands are shownon the left ordinate. Lane 1, untreated cells; lane 2, WR-1065-treatedbut unirradiated cells, lane 3; irradiated cells; lane 4, cellsirradiated and treated with WR-1065.

FIG. 10G results from immunoblot analysis of topo II levels in rapidlylysed cells. conditions were similar to those described in FIG. 10F withthe exception that cells were lysed in electrophoresis sample buffercontaining 2% SDS by boiling for 2 min.

FIGS. 10H and 10I show, respectively, topo I (FIG. 10G) and topo IIα(FIG. 10I) activity in cell-free extracts. Reaction mixtures wereassayed for topo I-mediated relaxation of pUC8 plasmid DNA and topoIIα-mediated unknotting of P4 phage DNA, as described herein: FIG. 10H,lane 1, pUC8 DNA only; lane 2, no drug; lane 3, 0.4 mM WR-1065; lane 4,4 mM WR-1065; lane 5, 40 mM WR-1065; lane 6, 0.5 mM Camptothecin. FIG.10I, lane 1, no drug; lane 2, 0.4 mM WR-1065; lane 3, 4 mM WR-1065; lane4, 40 mM WR-1065; lane 5, 0.3 mM Genestein.

FIG. 10J, FIG. 10K, FIG. 10L, FIG. 10M, FIG. 10N, FIG. 10O, FIG. 10P andFIG. 10Q show typical flow cytometry patterns describing the DNAdistribution of K1 cells exposed to 4 mM WR-1065 for 0 min, (FIG. 10J)30 min, (FIG. 10K) 1.0 hour (FIG. 10L), 2.0 hours (FIG. 10M), 3.0 hours(FIG. 10N), 4.0 hours (FIG. 10O), 5.0 hours (FIG. 10P) and 6.0 hours(FIG. 10Q). During the 6 h exposure, the percent of cells in G1 fellfrom 39 to 21, while the percent of cells in G2 increased from 18 to 27.The percent of cells in S ranged from 43 to 46.

FIGS. 11A-11D demonstrate the identity in mutations observed in bothmouse and human T-lymphocytes at the HPRT locus, upon treatment withcytoxan--as is also observed after irradiation. The anti-mitageniceffect of WR-2721 and/or its associated metabolites was demonstrated inmice treated with cytoxan or cisplatin. Each error bar is one standarderror of the mean.

FIG. 12A, FIG. 12B, FIG. 12C, FIG. 12D, FIG. 12E, FIG. 12F, FIG. 12G,FIG. 12H, FIG. 12I, FIG. 12J and FIG. 12K illustrate the anti-mutageniceffect of WR-33278 electroporated into CHO AA8 cells.

FIG. 12A and FIG. 12B. Effects of WR-33278 (open bar) and spermine(hatched bar) on CHO AA8 cell survival (FIG. 12A) and mutation inductionat the hprt locus (FIG. 12B). Drug-only bars represent the effects of0.01 mM WR-33278 or 0.01 mM spermine on these processes. FIG. 12A:compared with its corresponding drug exposure only group, all cellsurvivals in each of the electroporated groups are significantly reduced(student's two-tailed t test, P>0.001). FIG. 12B: compared with itscorresponding drug exposure only group, the number of mutants per 10⁶surviving cells in each of the electroporated groups is notsignificantly different (P>0.01). Data presented are from a minimum of 3replicate experiments. Error bars represent one standard of the mean.

FIGS. 12C and 12D. Effect of electroporation on radiation-induced cellkilling (FIG. 12C). and mutagenesis at the hprt locus (FIG. 12D). FIG.12C: as compared with cell killing radiation by radiation only, cellsurvival was significantly reduced by electroporation performed 30 minbefore (P=0.007) or 3 h after (P>0.001) irradiation. FIG. 12D: mutationinduction was significantly enhanced by electroporation performed 30 minbefore (P>0.001) or 3 h after (P>0.001) irradiation. Experiments wererepeated a minimum of 3 times. Error bars represent one standard errorof the mean.

FIG. 12E and FIG. 12F. Effect of electroporation with either WR-33278(open bars) or spermine (hatched bars) on the survival of cellsirradiated with 750 cGy either 30 min after (FIG. 12E) or 3 h before(FIG. 12F) electroporation. FIG. 12E: comparing electroporation with nodrug 30 min prior to irradiation, electroporation of 0.01 mM WR-33278 orspermine 30 min prior to irradiation significantly protected againstcell killing (P=0.006 and P=0.013, respectively). FIG. 12F: comparingelectroporation with no drug 3 h after irradiation, electroporation ofWR-33278 did not affect cell survival (0.01 mM, P=0.1; and 0.001 mM,P=0.1). Electroporation of spermine at a concentration of 0.01 mM wasmore effective (P=0.01) than a concentration of 0.001 mM (P=0.33). Allexperiments were repeated a minimum of 3 times. Error bars equal onestandard error of the mean.

FIG. 12G and FIG. 12H. Effect of electroporation with either WR-33278(open bars) or spermine (hatched bars) on hprt mutation induction incells irradiated with 750 cGy either 30 min after (FIG. 12G) or 3 hbefore (FIG. 12H) electroporation. FIG. 12G: comparing electroporationwith no drug 30 min prior to irradiation, electroporation of both 0.01mM and 0.001 mM WR-33278 or spermine were highly effective in protectingagainst the induction of hprt mutants (P>0.001, P=0.015, P>0.001,P=0.04, respectively). FIG. 12H: comparing electroporation with no drug3 h following irradiation, electroporation of both 0.01 mM and 0.001 mMWR-33278 or spermine were highly effective in protecting against theinduction of hprt mutants (all P values >0.001). All experiments wererepeated a minimum of 3 times. Error bars represent one standard errorof the mean.

FIG. 12I, FIG. 12J and FIG. 12K illustrate the role performed by thepresence of an amine functionality, as evidenced through a comparison of1-cysteine and N-acetylcysteine (FIG. 12I: 1-cysteine; MEC-41, 43; FIG.12J: N-acetyl-1-cysteine; MEC-40, 42; FIG. 12K: N-aceytl-1-cysteine;MEC-40). Each error bar is one standard error of the mean.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is concerned with four general areas: (1)phosphorothioates and associated metabolites, when administered tomammals (i.e., mice) following mutagen exposure (i.e., ionizingradiation including photon and fission-spectrum neutrons and chemicalmutagens such as cis-diaminedichloroplatinum (II) (cisplatin) andcytoxan),protect against genotoxic damage which normally leads to thedevelopment of somatic mutations--the same mutations observed in humanlymophocytes; (2) protection against mutagen-induced mutations by thephosphorothioates and associated metabolites at very low concentrationswhich are much less than required for protection against cell lethality;(3) protection against mutagen-induced somatic mutations by thephosphorothioates and associated metabolites, as shown to correlate mostclosely with the disulfide metabolite and the presence of a polyaminefunctionality; and (4) protection against mutagen-induced somaticmutations, as a general property of the genus of phosphorothioates andtheir associated metabolites irrespective of the origin of the mutagenicevent; all of which are demonstrated by the observed antimutagenicproperties of the species S-1-(aminoethyl) phosphorothioic acid(WR-638), S- 2-(3-methylaminopropyl) aminoethyl! phosphorothioate(WR3689), S-2-(4-aminobutylamino) ethylphosphorothioic acid (WR-2822),3- (2-mercapto ethyl) amino! propionamide p-toluenesulfonate (WR-2529),S-1-(2-hydroxy-3-amino) propyl phosphorothioic acid (WR-77913), 2-3-(methylamino) propylamino! ethanethiol WR-255591),S-2-(5-aminopentylamino) ethyl phosphorothioic acid (WR-2823), and 1-3-(3-aminopropyl) thiazolidin-2-yl!-Dgluco-1,2,3,4,5 pentane-pentoldihydrochloride (WR-255709).

I. Phosphorothioate Genus Protection After Irradiation

Chemicals of the phosphorothioate genus and associated metabolites canprotect against somatic mutations when administered to mammals followinga mutagen exposure. This conclusion is based on the observation thatS-2-(3-aminopropylamino) ethyl phosphorothioic acid, administered at adose of 400 mg/kg up to 3 h following neutron radiation exposure,affords substantial protection against radiation-induced mutations atthe hypoxanthine-guanine phosphoribosyl transferase locus in the Tlymphocytes of mice (see FIG. 1, ref. 10). The magnitude of protectionis unchanged regardless of whether the phosphorothioate was administered30 min before, immediately following (ie., within 10 min), or up to 3 hfollowing irradiation of the test animals.

The spontaneous mutant frequency of T lymphocytes from unirradiatedcontrol animals was stable and ranged from 9-10×10⁻⁷. Followingirradiation with 150 cGy of fission neutrons, the mutant frequencyincreased to 5.6×10⁻⁵ ±2.3×10⁻⁵ (1 standard error of the mean). Mutantfrequencies in animals administered S-2-(3-aminopropylamino)ethylphosphorothioic acid 30 min before, immediately after, or 3 hfollowing irradiation with 150 cGy of fission neutrons were 1.1×10⁻⁵±2.6×10⁻⁶, 1.0×10⁻⁵ ±1.3×10⁻⁶, and 1.4×10⁻⁵ +5.8×10⁻⁶, respectively.

As stated above, the aminothiol 2- (aminopropyl)amino!ethanethiol(WR-1065) is the active thiol ofS-2-(3-aminopropylamino)ethylphosphorotioic acid (WR-2721). Aminothiols,such as WR-1065 and its associated disulfide metabolite, are effectivein inhibiting DNA synthesis, strand rejoining, nuclease activity, andcell cycle progression in mammalian cells. These effects on cellularenzymatic processes indicate aminothiol protection against mutagenesisincludes modulation of endogenous enzyme processes relating to DNAsynthesis and repair. WR-1065 is an effective radiation protector andantimutagenic agent when it is administered 30 min prior to radiationexposure to Chinese hamster ovary K1 cells (i.e., a dose modificationfactor of 1.4) at a concentration of 4 mM. Under these exposureconditions, topoisomerase (topo) I and IIα activities and associatedprotein contents were measured in the K1 cell line using the DNArelaxation assay, the P4 unknotting assay, and immunoblotting,respectively. WR-1065 was ineffective in modifying topo I activity, butit did reduce topo II activity by an average of 50 percent. Themagnitude of topo IIα protein content, however, was not affected bythese exposure conditions. (See FIGS. 10A-FIG. 10I.) Cell cycle effectswere monitored by the method of flow cytometry. Exposure of cells to 4mM WR-1065 for a period of up to 6 h resulted in a buildup of cells inthe G2 compartment. (FIG. 10J-FIG. 10Q.) This observed cell cycle delayin conjunction with reduction in topo IIα activity indicates more timeavailable for the repair of cell damage and suggests genomestabilization and increased efficiency of repair processes.

These results demonstrate, in particular, a modifying effect by 2(aminopropyl)-amino!ethanethiol on type II topoisomerase, which isinvolved in DNA synthesis. In contrast to typical topo II inhibitorsused in chemotherapy, WR-1065 and/or its disulfide are effective agentsagainst both radiation-induced cell lethality and mutagenesis. Atconcentrations up to 40 mM, WR-1065 did not affect the activity ofeither topo II or topo IIα, as compared to inhibitors Camptothecin andGenistein, suggesting, without being bound to any one theory ormechanism of operation, that WR-1065-induced reduction in topo IIαactivity may be due to some indirect effect. Without limitation, thisobservation may involve inhibition of protein kinase C-mediatedmetabolic phosphorylation of topo IIα by WR-1065. Inhibitingphosphorylation could reduce the activity of enzymes that serve assubstrates for this protein kinase. This possible mode of action isconsistent with the observed reduction in the catalytic activity of topoIIα and WR-1065-treated K1 cells (determined by the unknotting assay),without a concomitant reduction of topo IIα protein levels (determinedby immunoblotting).

The topoisomerase studies demonstrate the ability of phosphorothioatesand associated metabolites to influence cellular response to mutagenicinsult and cellular enzymatic activities involved in DNA synthesis, cellcycle progression and, possibly, repair.

Referring to FIG. 11A and FIG. 11B, mice and human cancer patients,respectively, were treated with cytoxan and observed with respect to theincrease in mutant frequency. As shown, in comparison with untreatedpopulations, both the mouse and human subjects exhibited substantialcytoxan-induced mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus--consistent with the radiation-inducedmutagenesis, described above, and supporting the proposition that thesame mutation is observed irrespective of the nature and/or source ofthe mutagenic event. The mutant frequencies of mice T-lymphocytes weredetermined as described above. The human lymphocytes were obtained fromblood samples of patients after the cytoxan treatment, using standardcell stimulation techniques and hprt assays.

The anti-mutagenic effect of WR-2721 was demonstrated at the hprt locusin mice treated with cytoxan and cisplatin, FIG. 11C and FIG. 11D,respectively. The reduction in mutant frequencies of T-lymphocytesisolated from mice so treated shows WR-2721 and its metabolites to beeffective as an antimutagens against chemical as well as radiationinsult.

II. Phosphorothioate Protection from Low Dosages

The phosphorothioates and associated metabolites further achieve mutagenprotection at very low concentrations, compared to concentrationsrequired to protect against cell lethality. This conclusion is based onthe observations that S-2-(3-aminopropylamino) ethyl phosphorothioicacid is equally antimutagenic at concentrations of 400 mg/kg, 200 mg/kg,100 mg/kg, and 50 mg/kg (see FIG. 2, ref. 10). Mutant frequencies of Tlymphocytes isolated from mice irradiated with 150 cGy of fissionneutrons were 9.0×10⁻⁵ ±1.2×10⁻⁵ (1 standard error of the mean) forirradiated controls, 1.2×10⁻⁵ ±1.0×10⁻⁵ (S.E.) for 400 mg/kg, 7.8×10⁻⁶±2.7×10⁻⁶ (S.E.) for 200 mg/kg, 1.5×10⁻⁵ ±1.4×10⁻⁶ (S.E.) for 100 mg/kg,and 6.3×10⁻⁶ ±3.2×10⁻⁶ (S.E.) for 50 mg/kg. Under in vitro conditions,the free thiol form of S-2-(3-aminopropylamino) ethylphosphorothioicacid, i.e., 2- (aminopropyl) amino! ethanethiol was administered as anantimutagen to cultured Chinese hamster ovary cells at a concentrationrange from 4 mM down to 0.01 mM. When administered 30 min prior toirradiation with 750 cGy of ⁶⁰ Co gamma rays (see FIG. 3), the drug andits metabolite is significantly effective as an antimutagen.

Administration of 2- (aminopropyl) amino! ethanethiol also results inthe formation of its disulfide. Protection against the cell killingeffects of radiation by 2- (aminopropyl) amino! ethanethiol rapidlydiminishes as the concentration falls from 4 mM to 0.01 mM (see FIG. 4).

III. Disulfide Metabolite Mutagenic Protection

The presence of disulfide metabolite of the phosphorothioate class ofcompounds corresponds to antimutagenic protection. This conclusion isbased on the observations that, following the administration of 4 mM of2- (aminopropyl) amino! ethanethiol, protection againstradiation-induced (i.e., fission neutrons) somatic mutations at thehypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamsterovary cells correlates with the measured disulfide as compared to thefree thiol (see FIG. 5A, FIG. 5B and FIG. 5C).

Subsequent thiol and disulfide concentrations were measured by usingmonobromobiamine (mBBr), which reacts selectively with thiols via a Sn 2displacement process to produce a fluorescent derivative. These methodswere developed to specifically measure 2- (aminopropyl) amino!ethanethiol, its phosphorothioate, and its disulfide. Chinese hamsterovary cells, 5×10⁶ in 5 ml of growth medium, were administered 4 mM of2- (aminopropyl) amino! ethanethiol for 30 min at 37° C. They were thencentrifuged, washed with a buffer, and resuspended in fresh medium up toan additional 4 h. After 15 min, 30 min, 1 h, 2 h, and 4 h ofincubation, a sample of cells was removed and exposed to 150 cGy offission neutrons. At these times various measurements made included:survival measurements, mutation measurements, and intracelluarmeasurements of 2- (aminopropyl) amino! ethanethiol and its disulfide.The data contained in FIG. 5A, FIG. 5B and FIG. 5C demonstrate thatsurvival protection is well correlated with thiol measurements. This isconsistent with conventional understandings and teachings. The disulfideconcentration was measured to be significantly less than that of thethiol, but the rate of its decrease with time was less than that foundfor the thiol. Measured protection against mutagenesis remained constantover this time range correlating with the kinetics of disulfide asopposed to the thiol concentration. The disulfide form of this thiolclosely resembles the polyamine spermine (see FIG. 6A and FIG. 6B).Polyamines are known to be involved in the repair of DNA damage due toionizing and UV irradiation. The measurements indicate an inability toprotect against radiation-induced lethality by the phosphorothioateclass of chemicals and their associated metabolites when they are addedafter radiation. Coupling these data with the demonstrated ability toprotect against radiation-induced mutagenesis under similar postradiation exposure conditions, make it clear that it is thus thefidelity, not the amount or quantity, of DNA damage which is beingaffected by these agents. This is also consistent with the properties ofpolyamines which have been shown to stabilize DNA against enzymaticdegradation.

The prior art has indicated that the disulfide is not a protectivemetabolite of either the phosphorothioates or thiols. The instant dataindicates however that the disulfide metabolite of the phosphorothioateis a protective moiety in preventing mutagen- (i.e., radiation) inducedsomatic mutations. The disulfide metabolite has a close similarity instructure and composition to polyamines, which are known endogenousagents capable of stabilizing chromatin and affecting DNA repair.Further, the phosphorothioates S-2-(3-aminopropylamino) ethyl (WR-2721),S-2-(4-aminobutylamino) ethyl (WR-2822), and S-2-(7-aminoheptylamino)ethyl have been shown in the prior art to competitively inhibit theuptake of the polyamine putrescine into rat lung tissue. The importanceof the disulfide moiety in the post mutagen (i.e., radiation)exposure-protection process against the formation of somatic mutationsdemonstrates a surprising advantage for phosphorothioate compounds whichform polyamine-like disulfides for use as antimutagenic chemopreventiveagents.

The polyamine spermine and the disulfide WR-33278 are structurallysimilar agents capable of binding to DNA. As described above, WR-33278is the disulfide metabolite of theS-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721). Because oftheir reported structural and functional similarties, spermine andWR-33278 were compared with respect to cell survival and mutationinduction at the hypoxanthine-guanine phosphoribosyl transferase (hprt)locus in Chinese hamster AA8 cells. Both WR-33278 and spermine wereshown to be effective in protecting against radiation-inducedmutagenesis, whether administered before or after irradiation.

In order to facilitate both the uptake of WR-33278 into cells and thedirect comparison between the protective properties of WR-33278 andspermine, these agents (at concentrations of 0.01 mM and 0.001 mM) wereelectroporated into cells. Electroporation, 300 V and 125 μFD, wasperformed either 30 min prior to or 3 h following exposure of cells to750 cGy (⁶⁰ Co gamma rays) of ionizing radiation. Electroporation alonereduced cell survival to 75% but had no effect on hprt mutationfrequency. (See FIG. 12A, FIG. 12B, FIG. 12C and FIG. 12D.) Theelectroporation of either spermine or WR-33278 at concentrations greaterthan 0.01 mM was extremely toxic and, therefore, precluded the study ofhigher concentrations of these agents. The exposure of cells to bothelectroporation and irradiation gave rise to enhanced cell killing andmutation induction, with the sequence of irradiation followed 3 h laterby electroporation being the more toxic protocol. Cell survival valuesat a radiation dose of 750 cGy were enhanced by factors of 1.3 and 1.8following electroporation of 0.01 mM of spermine and WR-33278,respectively, 30 min prior to irradiation. Neither agent was protectiveat a concentration of 0.001 mM. (See FIG. 12E, FIG. 12F, FIG. 12G andFIG. 12H.

Protection against radiation-induced hprt mutations was observed forboth spermine and WR-33278 under all experimental conditions tested.Spermine at concentrations of 0.01 mM and 0.001 mM administered 30 minbefore or 3 h after irradiation reduced mutation frequencies by 2.2,1.2, 1.9 and 2.2, respectively. WR-33278 at concentrations of 0.01 mMand 0.001 mM administered 30 min before or 3 h after irradiation loweredmutation frequencies by factors of 1.8, 1.3, 1.4 and 2.0, respectively.

The close agreement in the magnitudes of effect induced by spermine andWR-33278 against mutagenesis is consistent with their known structuraland functional similarities. These data suggest that the properties ofradioprotection and chemoprevention exhibited by the phosphorothioate(WR-2721) and associated aminothiol (WR-1065) and disulfide (WR-33278)metabolites may be mediated in part via endogenous polyamine-likeprocesses. Such a mechanism has important implications with respect tothe design and development of a new generation drugs for use inradioprotective and chemopreventive agents.

To determine what role, if any, is performed by the amine functionalityin either the WR-2721, WR-1065, or WR-33278 anti-mutagens, the radiationsurvival, protection and anti-mutagenic properties of the aminothiols1-cysteine and N-acetylcysteine were compared. As shown in FIG. 12I,FIG. 12J and FIG. 12K 1-cysteine is an effective radioprotector,rendered less effective when the amino group is acetylated (FIGS. 12Iand FIG. 12J) Protection against radiation-induced mutagenesis at thehprt locus in CHO AA8 cells is also adversely affected, furthersupporting the proposition that, at least in part, an aminefunctionality present in conjunction a phosphorothioate, thiol, ordisulfide functionality may be responsible for protection againstmutagenicity by WR-2721 and its metabolites.

IV. Phosphorothioate Protection Against Mutagenesis

The ability to protect against mutagen-induced somatic mutations is ageneral property of the phosphorothioates and their associatedmetabolites. This advantage is demonstrated by the data obtained byexperiments on cultured Chinese hamster ovary cells first exposed to 150cGy of fission neutrons and then applying for 30 min a quantity of 4 mMof either 3- (2-mercaptoethyl) amino! propionamide p-toluenesulfonate(WR-2529), S-i-(aminoethyl) phosphorothioic acid (WR-638), S-2-(3-methyl aminopropyl) aminoethyl! phosphorothioate acid (WR-3689),and S-1-(2-hydroxy-3-amino) propyl phosphorothioic acid (WR-77913) (seeFIG. 8). All of these agents, including 2- 3-(methylamino) propylamino!ethanethiol (WR-255591) were effective anti-mutagens when they wereadded to cells at a concentration of 4 mM at about 30 min prior toexposure to fission neutrons (see FIG. 8). Protection againstradiation-induced somatic mutations in mammals (i.e., mice) was alsodemonstrated for S-1-(aminoethyl) phosphorothioic acid (WR-638) underconditions in which a dose of 520 mg/kg was administered ip to animalswithin about 10 min after whole-body exposure to 750 cGy of ⁶⁰ Co gammarays (see FIG. 9). Phosphorothioates exhibited antimutagenic propertiesin mammals when administered 30 min prior to exposure to 750 cGy of ⁶⁰Co gamma rays. The phosphorothioates included S- 2-(3-methylaminopropyl)aminoethyl! phosphorothioate acid (WR-3689), and S-2-(4-aminobutylamino)ethylphosphorothioic acid (WR-2822). These data demonstrate that theantimutagenic properties of S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721) are also observable in selected onesof the phosphorothioates and their associated metabolites.

While preferred embodiments of the invention have been shown anddescribed, it will be clear to those skilled in the art that variouschanges and modifications can be made without departing from theinvention in its broader aspects as set forth in the claims providedhereinafter.

What is claimed is:
 1. A method for reducing mammalian cell mutationsinduced by a mutagenic event, comprising the steps of:(a) preparing adosage of an effective amount of a chemical compound selected from thegroup consisting of an aminoalkylphosphorothioate and an associatedaminoalkylphosphorothioate metabolite; and (b) administering said dosageto a mammal at an effective time before or after exposure to saidmutagenic event.
 2. A method for reducing mammalian cell mutationsinduced by a genotoxic event, comprising the steps of:(a) preparing adosage of an effective amount of a chemical compound selected from thegroup consisting of an aminoalkylphosphorothioate and an associatedaminoalkylphosphorothioate metabolite; and (b) administering said dosageto a mammal in an amount and at a time effective to prevent loss of cellfunction and genetic information.
 3. The method of claim 1, wherein saidmutagenic event is induced by a chemotherapeutic agent.
 4. The method ofclaim 3, wherein said chemotherapeutic agent is cisplatin.
 5. The methodof claim 3, wherein said chemotherapeutic agent is cytoxan.
 6. Themethod of claim 1, wherein said dosage is an effective amount less thanabout 400 mg/kg of mammal body weight.
 7. The method of claim 6, whereinsaid dosage is an effective amount less than about 50 mg/kg of mammalbody weight.
 8. The method of claim 7, wherein said dosage is aneffective amount less than about 25 mg/kg of mammal body weight.
 9. Themethod of claim 1, wherein said dosage of said compound is administeredto a mammal at a time between about 30 minutes before and about 3 hoursafter said mutagenic event.
 10. The method of claim 9, wherein saiddosage of said compound is administered to said mammal at a time betweenabout 30 minutes before and the time of the mutagenic event.
 11. Themethod of claim 9, wherein said dosage of said compound is administeredto said mammal at a time between about the time of the mutagenic eventand about 3 hours after the time of the mutagenic event.
 12. The methodof claim 1, wherein said aminoalkylphosphorothioate or associatedmetabolite compound are selected from the group consisting ofS-1-(aminoethyl) phosphorothioic acid (WR-638), S-2-(3-methylaminopropyl) aminoethyl! phosphorothioate acid (WR-3689),S-2- 4-aminobutylamino) ethylphosphorothioic acid (WR-2822), 3-(2-mercapto ethyl) amino! propionamide p-toluenesulfonate (WR-2529),S-1-(2-hydroxy-3-amino) propyl phosphorothioic acid (WR-77913), 2-3-(methylamino) propylamino! ethanethiol (WR-255591),S-2-(5-aminopentylamino) ethyl phosphorothioic acid (WR-2823), and 1-3-(3- aminopropyl) thiazolidin-2-Y1!-D-gluco-1,2,3,4,5 pentane-pentoldihydrochloride (WR-255709).
 13. The method of claim 12, wherein saidcompound is S-1-(aminoethyl) phosphorothioic acid (WR-638).
 14. Themethod of claim 12, wherein said compound is S- 2-(3-methylaminopropyl)aminoethyl! phosphorothioate acid (WR-3689).
 15. The method of claim 12,wherein said compound is S-2- 4-aminobutylamino) ethylphosphorothioicacid (WR-2822).
 16. The method of claim 12, wherein said compound is 3-(2-mercapto ethyl) amino! propionamide p-toluenesulfonate (WR-2529). 17.The method of claim 12, wherein said compound is S-1-(2-hydroxy-3-amino)propyl phosphorothioic acid (WR-77913).
 18. The method of claim 12,wherein said compound is 2- 3-(methylamino) propylamino! ethanethiol(WR-255591).
 19. The method of claim 12, wherein said compound isS-2-(5-aminopentylamino) ethyl phosphorothioic acid (WR-2823).
 20. Themethod of claim 12, wherein said compound is 1-3(3-aminopropyl)thiazolidin-2Y1!-D-gluco-1,2,3,4,5 pentane-pentoldihydrochloride (WR-255709).
 21. The method of claim 1, wherein saidcompound is an aminoalkylphosphorothioate that forms a polyamine-likedisulfide under conditions of cellular metabolism.
 22. The method ofclaim 1, wherein said chemical compound is a metabolite of anaminoalkylphosphorothioate compound.
 23. The method of claim 22, whereinsaid metabolite of said compound forms a polyamine compound whenadministered to the mammal.
 24. The method of claim 22, wherein saidmetabolite of said compound isN,N,'-dithiodi-2,1-(ethanediyl)bis-1,3-propanediamine (WR-33278).